Process for production of L-lysine by fermentation

ABSTRACT

A process for the production of L-lysine by fermentation which comprises cultivating aerobically an L-lysine-producing mutant strain of a microorganism belonging to the Genus Pseudomonas or Achromobacter in a culture medium containing a hydrocarbon as a main carbon source until a substantial amount of L-lysine is accumulated in the culture medium and recovering the thus accumulated L-lysine from the culture broth is disclosed. The mutant strain used in the present invention has a resistance to at least one amino acid or analogue thereof selected from the group consisting of L-valine, Alpha -aminobutyric acid, norvaline, +-hydroxynorvaline, Alpha -amino- Beta -chlorobutyric acid and L-threonine.

United States Patent [191 Watanabe et al.

[ PROCESS FOR PRODUCTION OF L-LYSINE BY FERMENTATION [76] Inventors: Kiyoshi Watanabe; Tutomu Tanaka;

Tamotsu Hirakawa; Hideaki Kinoshita; Mamoru Sasaki; Koji Obayashi, all of Hyogo, Japan [73] Assignee: Kanegafuchi Chemical Industries,

Osaka, Japan [22] Filed: Aug. 2, 1973 21 Appl. No.: 384,844

[30] Foreign Application Priority Data Aug, 2, 1972 Japan 47-77978 Aug 2, I972 Japan 4717979 {52] US. Cl 195/28 R; l95/47; [95/112 [5]] Int. Cl Cl2b [/00 [58} Field of Search l95/28 R, 29, 47 H2 (56] References Cited UNITED STATES PATENTS 1756,9l6 lU/l973 Leuvitt .i 195/47 [4 1 Sept. 16, 1975 Zinn & Macpeak i 1 ABSTRACT A process for the production of L lysine by fermentation which comprises cultivating aerobically an L- lysine-producing mutant strain of a microorganism belonging to the Genus Pseudomonas or Achromobacter in a culture medium containing a hydrocarbon as a main carbon source until a substantial amount of L- Iysine is accumulated in the culture medium and re covering the thus accumulated L-lysine from the culture broth is disclosed. The mutant strain used in the present invention has a resistance to at least one amino acid or analogue thereof selected from the group consisting of L-valine, waminobutyric acid, norvaline, B-hydroxynorvaline, a-amino-B-chlorobutyric acid and L-threonine.

5 Claims, N0 Drawings isolating the thus obtained mutant, i.e.. L-valine-, L- PROCESS FOR PRODUCTION OF L-LYSINE BY threnine-, L-valine analogueor L-threonine ana- FERMENTATION logue -resistant strain and culturing the isolated strain BACKGROUND OF THE INVENTION "aerobically in a culture medium containing a hydrocar- 5 bon as a mian carbon source until a substantial amount I. FIELD OF THE INVENTION of L-lysine is accumulated in the culture broth.

This invention relates to a process for producing L- As described above, it is known that some strains belysine from hydrocarbons by fermentation using a mi' longing .to the Genus Pseudomonas and the Genus croorganism. More particularly, this invention relates Achromobacter produce a small amount of L-lysine. to a process for producing L-lysine which comprises Contrary to the prior art, in accordance with the presaerobieally culturing an L-lysine-producing microorent invention, a strain which is capable of producing ganism having a high resistance to L-valine, L-threo- L-lysine in a high yield is isolated on the basis of the nine or animo acid analogues of L-valine or L-threo finding, e.g., a correlation between L-valine-sensitivity nine selected from the mutants belonging to the Genus and L-lysine productivity, and the thus isolated strain is Pseudomonas and the Genus Achromobacter in a cul- I5 greatly improved in the L-lysine productivity. ture medium containing a hydrocarbon as a main car- That is, the present inventors found that when Pseubon source until a substantial amount of L-lysine is acdomonas brevis is cultured using ethanol as a carbon cumulated in the culture medium and recovering the source, this strain exhibits a valine-sensitivity, i.e., the thus accumulated L-lysine from the culture broth. growth of Pseudomonas brevis is extremely inhibited in 2. DESCRlPTlON OF THE PRIOR ART the presence of L-valine. Further, they found that the It is well-known that L-lysine is one of the important accumulation of L-lysine can be increased by isolating essential amino acids and the demand for producing L- a novel strain having a weak L-valine sensitivity, i.e., an lysine inexpensively has steadily been increased partic- L-valine-resistant strain obtained by artificial mutation ularly from the standpoint of use for the supplements or and aerobically culturing the L-valine-resistant strain additives to cereals as main diets for human being. using a hydrocarbon as a carbon source. That is, these Various processes have been proposed for producing findings indicate that L-lysine productivity can be in- L-lysine by fermentation, and some processes utilizing creased and improved as the valine-resistance of the inexpensive hydrocarbons as carbon sources have above strain increases. On the basis of these findings, beeen described in publications (for example, British the present inventors have established a novel tech- Pat. Nos, l,l84,530, l,l86,989 and l,24l,90l, etc.). nique which makes it possible to find out the microor- However, these well-known processes are disadvantaganisms having an ability to produce L-lysine in high gcous in that the yield of the desired L-lysine is low and yields by isolating the strains which are resistant to an therefore processes which are advantageous in indusamino acid such as L-valine or L-threoninc, or to a so trial production of L-lysine have not yet been estabcalled amino acid-analogue known as L-valineor L- lished. threonine-analogue, i.e., a-amino-n-butyrie acid, a-

lt is also known that some microorganisms belonging amino-B-chlorobutyric acid, norvaline, B-hydroxynorto the Genus Pseudomonas (French Pat. No. valine or the like.

2,041,650) or the Genus Achromobacter [Agr. Biol, As described previously, it is well known that certain Chem. 27 l l pp 773783 l963)] produce L-lysine microorganisms requiring homoserine as a growth facfrom hydrocarbons. However, it is very difficult to mi tor produce L-lysine by fermentation using carbohylize these known microorganisms in the industrial prodrates as carbon sources (German Pat. No. 2,034,406). duction of L-Iysine since the accumulated amount of However, the present invention is directed to the pro- L-lysine is extremely low, e.g., on the order of 0.3 to duction of L-lysine comprising culturing an L-lysinel.6 g/l. producing microorganism belonging to the Genus Further, it is known that L lysine can be produced Pseudomonas and the Genus Achromobacter which using a microorganism which is resistant to threonine, utilizes hydrocarbons and does not require any nutrient a amino-B-hydroxyvaleric acid, norleucine. aas a growth factor. aminobutyric acid or the like (German Pat. No. The microorganisms used in the present invention 2,034,406), but this process utilizes one ofhomoserineare newly isolated from the soil by the present invenrequiring microorganisms (Genus Brevibacterium, tors, and, upon comparison of their microbiological Genus Corynebacterium, Genus Arthrobacter, Genus properties in accordance with the criteria given in Ber- Microbacterium and Genus Nocardia) and carbohygeys Manual of Determinative Bacteriology, 7th Edidrates as carbon sources. tion, none of the microorganisms used in the present invention has conformity with the well-known microor- SUMMARY OF THE INVENTION ganisms. Thus, the microorganisms used in the present As a result of extensive studies on the isolation of varinvention are considered to be novel strains and desig ious microorganisms having a high ability to utilize hynated as Pseudomonas brevis No. 22 (ATCC 21940) drocarbons from the soil followed by screening of the and Ac'liromobacler cougulans No. 42 (ATCC 21934) isolated microorganisms, the present inventors unexand are now deposited at the American Type Culture pectedly found that a significant amount of L-lysinc can be accumulated in the culture broth by subjecting the bacteria belonging to the Genus Pseudomonas and the Genus Achromobacter to a mutational treatment.

Pseudomonas hrcvis Collection under the assigned numbers as described above.

The microbiological properties of Pseudomonas brewlr and Arhmmobac'ter cougularzs are as follows:

Achromobactcr coagulans I. Microscopic Observation short rods having round short ends; having round Pseudomonas brevis -continued Achromobacter coagulans ends; size: 0.7 1.0 X

(4) Stick Culture on Nutrient Gelatin no liquefaction brown soluble pigment growth on surface (5) Litmus Milk acidic coagulation lll. Physiological Properties l Nitrate Reduction (synthetic medium) positive (3) VP Reaction negative (4) lndolc Production negative (5) Hydrogen Sulfide Production negative to weak positive (6) Starch Hydrolysis negative (7) Citric Acid Utilization positive (ll) Urcasc positive (9] Cata'lase positive (10) Acid Production from Sugars produced from xylose. glucose. mannose and galactose within l0 days; produced from lactose within 30 days; only slightly produced from arabinose and fructose; no production from maltose. sucrose. trehalosc, sorbitol. mannitol. inositol. glycerol and starch (l l Utilization of Carbon Sources growth in C C n-parafi'ln growth in acetic acid. citric acid. succinic acid. ethanol and propanol l2) Nutrient Requirements none l3) Optimum Growth Temperature 33C l4] Growahle pH Values 5.5 9.0 l5) aerobic (for oxygen) IV. Isolation Source soil rods; size: 0.8 l.(l X

no movement no spore formation acid-fast stain: negative Gram stain: negative round colony; complete margin milky white. glossy and smooth surface linear milky white glossy and smooth surface sometimes thin membrane on surface; sometimes granular growth; precipitate formed liquefaction growth on surface weak alkaline to neutral coagulation and decolorization positive negative negative weak positive negative positive negative positive no production from arabinosc, fructom. xylose. glucose. mannose. lactose. maltose. sucrose. trehalose. sorbitol. mannitol. inositol.

glycerol. starch and galactose growth in acetic acid. succinic acid. citric acid and ethanol 5.5 9.0 aerobic (for oxygen l soil According to the present invention. L-lysine can be produced advantageously by isolating the above described amino acid analogue-resistant or amino acidresistant strain after mutational treatment of microorganism of the Genus Pseudomonas or the Genus Achromobacter and culturing the thus isolated strain in a culture medium containing a hydrocarbon as a carbon source. Two or more amino acids or amino acid analogues may be used to produce a strain which is resistant to the amino compound used. In addition. it is also possible to effect the mutational treatments repcatedlyusing two or more amino acids or analogues thereof alternately.

The mutational treatment may be carried out by the conventional chemical or physical treatments commonly employed in producing mutants, for example. by using the well-known mutagenic agents such as N- methyl-N'-nitro-N-nitrosoguanidine. or the physical mutational treatment such as irradiation with ultraviolet rays, radio-active rays or other procedures.

A typical procedure for producing an L-valine-resistant strain comprises culturing a mutant obtained by a usual mutational treatment from a strain of the Genus Pseudomonas or Achromobacter in a minimum culture medium containing L-valine and ethanol (preferably 0.5 to 1.0% by weight of ethanol based on the total weight of the medium) as a carbon source for a period of 3 to 5 days to obtain a culture enriched with L- valine-resistant strain. A cell suspension thus prepared which is previously diluted with a physiological saline solution to an appropriate cell concentration is then spread onto a minimal medium, an agar plate, containing L-valine or aamino-butyric acid at a concentration more than 0.5 mg/ (using 0.5 to 1.0% ethanol as a carbon source) followed by being cultivated. Of colonies produced after 3 to 5 days cultivation, relatively large colonies are harvested. Alternatively, it is also possible to replace L-valine used above by the above-described various analogues or L-threonine at a concentration more than 0.5 mg/ml for the enrichment culture. It is also possible to culture the mutationally treated strain by directly streaking on a minimal medium, an agar plate containing above amino acids or amino acid analogues, These strains were often found to have an increased L-valine-resistance.

It is generally important to increase the L-valineresistance of the mutant stepwise by subjecting the mutant to the above mutational treatments repeatedly. In producing a resistant strain it is generally necessary to predetermine the optimum culture conditions in order to impart to the microorganism the strongest sensitivity to amino acids or amino acid analogues used for the mutational treatment. For this purpose, the selection of a carbon source to be used in the culture medium and the combination of various amino acids or amino acid analogues (two or more agents) should carefully be investigated. To this effect, the most preferably carbon source for L-valine-resistant strain is found to be ethanol. v

In carrying out the L-lysine production in accordance with the present invention, an L-valine-resistant strain is aerobically cultured by the well-known culturing technique in the culture medium containing a hydrocarbon as a main carbon source, a nitrogen source, inorganic salts and other additives.

Hydrocarbons which can preferably be used in the culture medium for lysine production of this invention are n-paraffins containing to carbon, preferably 13 to 18, atoms or kerosene.

Nitrogen sources which can be used in the culture medium include organic and inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium acetate, ammonium citrate, ammonium succinate and the like, urea and ammonia.

Inorganic salts used in the present invention include potassium phosphate, magnesium sulfate, manganese sulfate, zinc sulfate copper sulfate, ferrous sulfate, calcium carbonate and the like and added at a concentration commonly employed.

In addition, surface active agents, for example, polyoxycthylene sorbitan monoor trioleate (Tween 80 or,

85, manufactured by AtlasPowder Co., U.S.A.) may be effectively used in the culture medium in an amount of approximately 0.02 to 0.5% by weight based on the total amount of the culture medium,

The pH value of the culture medium is preferably maintained in the range of from about 6 to about 9,

6 preferably 6.5 to 8.0, during the whole period of cultivationr The cultivation is usually carried out at a temperature of from about 25 to about 40C, preferably 30 to 35C.

It is necessary to conduct the cultivation under an aerobic condition, for example, by stirring with aeration and/or shake-culturing.

Upon completion of the cultivation, the resulting microbial cells are removed from the culture broth by the well-known procedure such as filtration or centrifugation. The removal of the microbial cells can easily be conducted by heating the culture broth, for example, at a temperature of'from to C for a period of from 10 to 30 minutes and removing the microbial cells, although the heating is not essential. The desired L-lysine can be obtained from the filtrate or the supernatant in the form of L-lysine hydrochloride in accordance with the well-known procedure using an ion-exchange resin such as Amberlite IRC-SO, Amberlite IR- 20 (available from Rohm & Haas Co.) and the like.

Alternatively, a powder enriched in L-lysine can be obtained by extracting the culture broth or a filtrate (or a supernatant) obtained for the culture broth with a solvent such as n-hexane to extract any residual hydrocarbon used as a carbon source, concentrating the resulting broth under reduced pressure with or without removal of the microbial cells by filtration and finally drying the resulting concentrate by drumdrying, spraydrying and the like.

The present invention is further illustrated by the following examples but they are not to be construed as limiting the scope of this invention.

EXAMPLE 1 Pseudomonas brevis No. 22 (ATCC 21940) was treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by being cultivated in a culture medium containing L-valine or DL-a-amino-n-butyric acid at a concen tration more than 0.5 mg/ml. By repeating mutational treatments and increasing the concentration of L- valinc or DL-a-amino-n-butyric acid, the L-valineresistant strains No. 73 and No. 56 derived from Pseudumonas brevis No. 22 (ATCC 21940) were isolated from minimal agar plates which contained L-valine or DL-a-amino-n-butyric acid.

Each of Pseudumonas brevis strains No. 22 (ATCC 21940), No. 73 and No. 56 (ATCC 21941) was cultivated on a bouillon slant medium at a temperature of 33C overnight, A platinum loopful amount of each of the cultures was inoculated in a culture medium for L- lysine production having the following composition and then shake-cultured at a temperature of 33C for 7 days.

After completion of cultivation, the amount of the accumulated L-lysine was quantitatively determined by the conventional method. The results observed in the Lwaline-sensitivity test for the parent strain Pseudunmnus brevis No. 22 (ATCC 21940) and mutants Pseudo- P7207105 brevis No. 73 and Pseudomonus brevis No. 56 (ATCC 21941), and the accumulated amount of L- lysine in each case are shown in Table 1 below.

As is apparent from the results shown in Table I, it was found that the increase in L-valine-resistance would result in the remarkable increase in the productivity of L-lysine.

Table 1 Growth" Strain A Strain B" Slruin C I8 48 i8 48 1B 48 (hrs) -y/m| L-Va1ine i Valine- Resist- 50 ance Test" 100 200 lOOOO L-Valine-Resistance I00 -y/ml 200 'ylml 5000 'y/ml Amount of L-Lysine (Hydrochloride) Accumulated in 0.6 8.4 25.0 Medium After 7 Days Cultivation (g/l)" Note: I Growth. Growth I Slight growth No growth (2) Strain A: Pseudomonas brevis No. 22 (ATCC 2l940) Strain B: Pseudomonas brevis No. 73 Strain C: Pseudornonas brevis No. 56 (ATCC 2l94l (3) L-Valine-Resistance Test:

(Medium) 75% phosphoric acid 12 ml 4)z 4 6 g NaCl l g MgSO ,.7H,O 0.2 g CaCl,.2H O 0.1 g FeSO,.7H.,O 0.l g zns0,.7H,0 0.03 g MnSO .4H O 0.002 g KOH 14 g pure water [000 ml pH 7.0

10 ml. portions of the above medium were poured into large test tubes and sterilized. To each of the tubes was added 0.1 ml of 60% ethanol as a carbon source to an ethanol concentration of about 0.6%. Each medium was inoculated with a suspension of each strain (which had been cultured for 24 hours on a slant medium) in such an amount that the strain concentration in the medium would be 10 10 cells/ml, and then shake-cul' tured at a temperature of 33C. The L-valine-resistance was referred to as the maximum L-valine concentration at which the strain is growable.

(4) Medium for Lysine Production:

manufactured by Atlas Powder Cu.. U.S.A. ml portions of the above medium were poured into 500 ml shake-flasks and then sterilized u! |Zl|C fur l5 minutes. Calcium carbonate was added to the medium after sterilivcd separate-I EXAMPLE 2 Ac'hromobacler coagulans N0. 42 (ATCC 2l934) was treated with a mutagenic agent. N-methyl-N-nitro-N- nitrosoguanidinc. to obtain Aclzmnmbacter coagulum No. 36 (ATCC 2l935) which is resistant to DL-norvaline. The resulting strain was then cultivated on a bouillon slant medium overnight. A platinum loopful amount of the culture was then inoculated in a fermentation medium having the following composition and shake-cultured at a temperature of 33C for 7 days. Quantitative determination of L-lysine indicated that L-lysine hydrochloride was accumulated in the medium in an amount of 4.8 g/l for Aclrmmobacler coagulans No. 36 (ATCC 21935) whereas the amount of L-lysine hydrochloride accumulated by the parent strain, Achromobacter coagulans No. 42 (ATCC 21934), was found to be 0.5 g/l:

Medium for Lysine Fermentation p "manufactured h Atlas Powder (u. USA. (31'0 was added in the medium alter sterilized separately.

30ml portion of the above medium was poured into a 500 ml shake-flask and sterilized at a temperature of l20C for 15 minutes.

EXAMPLE 3 as described in Example 2 at a temperature of 33C for 7 days. The amount of L-lysine hydrochloride accumulated was found to be 3.2 g/l for Ac/cmmobacter (eagle-- ans No. l7 and [0.4 g/l for Ac/zrumubactcr conga/mu No.49 (ATCC 2 1936). whereas the amount of L-lysine hydrochloride accumulated for A. coagulans No. 42 (ATCC 21934) and Ac/zrumobacler cuuguluns No. 36 (ATCC 2l935) were found to be 0.4 g/l and 4.2 g/l. re spectively.

The above results indicate that the resistance to norvaline and DL-a-amino-n-butyric acid can be increased by using these compounds alternately so that the lysine productivity of these microorganisms can be increased stepwise.

EXAMPLE 4 Valine-rcsistant strain. Pseudmnonas brevis No. 56 (ATCC 2l94l which had been obtained from Pseudr monus brews- No. 22 was cultivated on a bouillon agar slant medium at a temperature of 33C overnight and then inoculated in a 2 l shake-flask containing 600 ml of the previously sterilized seed culture medium having the following composition followed by being shake-cub tured at a temperature of 33C for 24 hours:

Secil (u lture Medium:

600 ml portion of the above seed culture was inoculatcd in a jar fermentor containing 20 l of a sterilized fermentation medium having the following composition and the inoculated culture was cultivated at a tem perature of 35C by agitating at a rate of 800 rpm with aeration at a rate of 27 l/min.

Fermentation Medium l 0 acid and then an alcohol were added thereto to obtain crystals weighing 35.3 g containing L-lysine hydrochloride having a purity more than 98% from i [of the culture broth.

EXAMPLE 5 A bouillon agar medium poured into a Houle bottle and then sterilized was inoculated with Ac/zrunwbuclcr coagm'uns No. 49 (ATCC 21936) which had been cultivated on a slant medium. After the inoculated microorganism was cultured at a temperature of 33C overnight, the culture was suspended in 300 ml of a sterilized physiological saline solution. 600 ml of the thus prepared suspension was inoculated into a ljar fermentor containing 15 l of the medium having the same composition as described in Example 2 and cultivated at a temperature of C with stirring at a rate of 500 rpm under aeration at a rate of 27 l/min. The pH value of the culture during the cultivation was adjusted to near neutral with an aqueous ammonia solution or hydrochloric acid. The amount of L-lysine hydrochloride accumulated after hours cultivation reached 13.4 g/l.

l I of the culture broth was centrifuged in a usual manner to remove microbial cells and L-lysine was adsorbed onto an ion-exchange resin (Amberlite lRC-SO) and then eluted with an aqueous ammonia solution. The eluate was concentrated, and hydrochloric acid and then an alcohol were added thereto to obtain l().2 g of crystals of L-lysinc hydrochloride.

While the invention has been described in detail and with reference to specific embodiments thereof. it will be apparent to one skilled in the art that various changed and modifications can be made therein with out departing from the spirit and scope thereof.

What is claimed is:

l. A process for the production of L-lysine by fermentation which comprises cultivating aerobically an L-lysine-producing mutant strain of a microorganism belonging to the Genus Pseudomonas or belonging to Acfimmobucter conga/uni ATCC 21934 which are the parent strains, in a culture medium containing a hydrocarbon as a main carbon source until a substantial amount of L lysine is accumulated in the culture medium and recovering the thus accumulated L-lysine from the culture broth. said mutant strain being resistant to at least one amino acid or analogue thereof selected from the group consisting of L-valine a-aminobutyric acid. norvaline. ;3-hydroxynorvaline. mammo- Bchlorobutyric acid and L-threonine.

2. A process according to claim 1 wherein said microorganism is a mutant strain of Pseudommmx brcvis ATCC 21940.

3. A process according to claim 1 wherein said mutant strain is Pscudomunus hrevir ATCC 2194i.

4. A process according to claim 1 wherein said mutant strain is A('hmnmbm'mr crmgnluns ATCC 2 l 935 or A'I'CC. 21936.

S. A process according to claim 1 wherein said by drocarbon is kerosene or nparaffin having l0 to 20 carbon atoms. 

1. A PROCESS FOR THE PRODUCTION OF L-LYSINE BY FERMENTATION WHICH COMPRISES CULTIVATING AEROBICALLY AN L-LYSINE-PRODUCING MUTANT STRAIN OF A MICROORGANISM BELONGING TO THE GENUS PSEUDOMONAS OR BELONGING TO ACHROMOBACTER COAGULANS ATCC 21934 WHICH ARE THE PARENT STRAINS, IN A CULTURE MEDIUM CONTAINING A HYDROCARBON AS A MAIN CARBON SOURCE UNTIL A SUBSTANTIAL AMOUNT OF L-LYSINE IS ACCUMULATED IN THE CULTURE MEDIUM AND RECOVERING THE THUS ACCUMULATED L-LYSINE FROM THE CULTURE BROTH, SAID MUTANT STRAIN BEING RESISTANT TO AT LEAST ONE AMINO ACID OR ANALOGUE THEREOF SELECTED FROM THE GROUP CONSISTING OF L-VALINE A-AMINO-BUTYRIC ACID, NORVALINE, BHYDROXYNORVALINE, A-AMINO-B-CHLOROBUTYRIC ACID AND L-THREONINE.
 2. A process according to claim 1 wherein said microorganism is a mutant strain of Pseudomonas brevis ATCC
 21940. 3. A process according to claim 1 wherein said mutant strain is Pseudomonas brevis ATCC
 21941. 4. A process according to claim 1 wherein said mutant strain is Achromobacter coagulans ATCC 21935 or ATCC
 21936. 5. A process according to claim 1 wherein said hydrocarbon is kerosene or n-paraffin having 10 to 20 carbon atoms. 